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The human reference genome represents only a small number of individuals, which limits its usefulness for genotyping. We present a method named HISAT2 (hierarchical indexing for spliced alignment of transcripts 2) that can align both DNA and RNA sequences using a graph Ferragina Manzini index. We use HISAT2 to represent and search an expanded model of the human reference genome in which over 14.5 million genomic variants in combination with haplotypes are incorporated into the data structure used for searching and alignment. We benchmark HISAT2 using simulated and real datasets to demonstrate that our strategy of representing a population of genomes, together with a fast, memory-efficient search algorithm, provides more detailed and accurate variant analyses than other methods. We apply HISAT2 for HLA typing and DNA fingerprinting; both applications form part of the HISAT-genotype software that enables analysis of haplotype-resolved genes or genomic regions. HISAT-genotype outperforms other computational methods and matches or exceeds the performance of laboratory-based assays. A graph-based genome indexing scheme enables variant-aware alignment of sequences with very low memory requirements.

Analysis of sleep for the diagnosis of sleep disorders such as Type-1 Narcolepsy (T1N) currently requires visual inspection of polysomnography records by trained scoring technicians. Here, we used neural networks in approximately 3,000 normal and abnormal sleep recordings to automate sleep stage scoring, producing a hypnodensity graph—a probability distribution conveying more information than classical hypnograms. Accuracy of sleep stage scoring was validated in 70 subjects assessed by six scorers. The best model performed better than any individual scorer (87% versus consensus). It also reliably scores sleep down to 5 s instead of 30 s scoring epochs. A T1N marker based on unusual sleep stage overlaps achieved a specificity of 96% and a sensitivity of 91%, validated in independent datasets. Addition of HLA-DQB1*06:02 typing increased specificity to 99%. Our method can reduce time spent in sleep clinics and automates T1N diagnosis. It also opens the possibility of diagnosing T1N using home sleep studies. The diagnosis of sleep disorders such as narcolepsy and insomnia currently requires experts to interpret sleep recordings (polysomnography). Here, the authors introduce a neural network analysis method for polysomnography that could reduce time spent in sleep clinics and automate narcolepsy diagnosis.

HLA class I glycoproteins contain the functional sites that bind peptide antigens and engage lymphocyte receptors. Recently, clinical application of sequence-based HLA typing has uncovered an unprecedented number of novel HLA class I alleles. Here we define the nature and extent of the variation in 3,489 HLA-A, 4,356 HLA-B and 3,111 HLA-C alleles. This analysis required development of suites of methods, having general applicability, for comparing and analyzing large numbers of homologous sequences. At least three amino-acid substitutions are present at every position in the polymorphic α1 and α2 domains of HLA-A, -B and -C. A minority of positions have an incidence >1% for the 'second' most frequent nucleotide, comprising 70 positions in HLA-A, 85 in HLA-B and 54 in HLA-C. The majority of these positions have three or four alternative nucleotides. These positions were subject to positive selection and correspond to binding sites for peptides and receptors. Most alleles of HLA class I (>80%) are very rare, often identified in one person or family, and they differ by point mutation from older, more common alleles. These alleles with single nucleotide polymorphisms reflect the germ-line mutation rate. Their frequency predicts the human population harbors 8-9 million HLA class I variants. The common alleles of human populations comprise 42 core alleles, which represent all selected polymorphism, and recombinants that have assorted this polymorphism.

In a comparison of tumors from patients with melanoma who benefitted from blockade of cytotoxic T-lymphocyte antigen 4 (CTLA-4) with tumors from patients who did not benefit, tumor neoantigens were detected that were strongly associated with a response. Immune checkpoint blockade has led to durable antitumor effects in patients with metastatic melanoma, non–small-cell lung cancer, and other tumor types, but the factors determining whether a patient will have a response remain elusive. 1 , 2 The fully human monoclonal antibodies ipilimumab and tremelimumab block cytotoxic T-lymphocyte antigen 4 (CTLA-4), resulting in T-cell activation. Some studies have established correlations between outcomes with ipilimumab and peripheral-blood lymphocyte count, markers of T-cell activation, 3 an “inflammatory” microenvironment, 4 , 5 and maintenance of high-frequency T-cell receptor clonotypes. 6 The relationship among the genomic landscape of the tumor, the mutational load, and the benefit from treatment remains obscure. . . .

IntroductionEmerging evidence supports a no-biopsy diagnosis for coeliac disease in patients with anti-tissue transglutaminase (tTG) titres ≥10 times the upper limit of normal (ULN). We investigated the diagnostic yield of tTG at thresholds below 10xULN and its performance characteristics in relation to clinical presentation, age, HLA genotype, total IgA, and endomysial antibodies (EMA).MethodsA retrospective analysis of a prospectively recruited cohort of adult patients (≥16 years) referred with suspected coeliac disease to a tertiary gastroenterology unit between 2015 and 2024. Patients with IgA deficiency or on a gluten-free diet at the time of gastroscopy were excluded. All patients had serum tTG, total IgA, and EMA measured. The time interval between serological testing and gastroscopy was within six weeks.ResultsA cohort of 1073 patients (median age 38 years (IQR 26-54), 67% female) met the study criteria. In total, 62% were diagnosed with coeliac disease (positive serology and Marsh 3), 15% had positive serology but Marsh 0-2 changes (potential coeliac disease), and 23% had negative serology and normal biopsies.Serum tTG ULN showed excellent diagnostic accuracy for the prediction of duodenal villous atrophy (AUC: 0.91, 95% CI: 0.89-0.93). The optimal tTG titre cut-off was ≥7xULN, demonstrating 96% specificity (95% CI: 93%-98%), 52% sensitivity (95% CI: 48%-56%), and 95% PPV (95% CI: 93%-97%). EMA testing did not provide any diagnostic advantage over a tTG titre cut-off of ≥7xULN. In patients with a false positive tTG at ≥7xULN, EMA was also falsely positive. In cases of a positive EMA result, the test showed lower specificity (90%) and PPV (92%), but higher sensitivity (67%).In patients with a high total IgA, applying a tTG titre cut-off of ≥7xULN resulted in a decrease in both specificity and PPV to 94%. Among 835 patients with HLA typing, individuals with DQ2 or DQ8 heterozygous genotype exhibited significantly lower tTG compared to homozygous individuals (median 2.9xULN (IQR 1.7-18.3) vs. 6.6xULN (IQR 0.8-10.1), p<0.001). Patients presenting with micronutrient deficiencies (iron, folate, vitamin B12, vitamin D) had significantly higher tTG (median 5.7xULN (IQR 1.4-18.3)) compared to those presenting with classical (median 2.7xULN (IQR 0.3-9.6), p<0.001) and non-classical (median 1.7xULN (IQR 0.2-8.1), p<0.001) symptoms of coeliac disease. There was a weak inverse relationship between tTG ULN and age (Spearman’s Rho -0.166, p<0.001).ConclusionThis is the largest study to date investigating the clinical utility of tTG in a no-biopsy context for coeliac disease. A tTG titre cut-off of ≥7xULN is a feasible diagnostic threshold. The role of total IgA extends beyond identifying those with a deficiency and demands careful evaluation. EMA testing offers limited diagnostic benefit over tTG in a no-biopsy approach.

Abstract HLA typing is crucial for assessing immunological compatibility between donors and recipients before transplantation. Deceased donor (DD) typing, typically via real-time PCR (rtPCR), provides rapid results vital for organ allocation. rtPCR primarily targets exons encoding the highly polymorphic peptide binding region (PBR) of HLA molecules. rtPCR cannot target all polymorphic sequences. Consequently, several possibilities of allele combinations are provided, leading to ambiguous typing results. As recent evidence suggests antibody responses can target regions in exons outside the PBR, notably in Exon 3 of the DPB1 locus, typing ambiguities can lead to the inability to assess histocompatibility between a donor and recipient. Herein, the aim was to investigate the utility of Next Generation Sequencing (NGS) in resolving DPB1 ambiguities in DD typing. A retrospective analysis of DD typing from February 2022 to January 2024 examined instances where NGS was used to resolve ambiguities reported via rtPCR. Each case was reviewed to understand the reason for resorting to NGS typing. The study focused on DPB1 ambiguities and assessed whether NGS effectively reduced ambiguity and differentiated alleles with polymorphisms outside the PBR. In total, 29 DD cases were found to have NGS testing results. In 14 cases, NGS was specifically performed to resolve DPB1 ambiguities. The ambiguities were significantly reduced from hundreds detected in rtPCR to 4 or fewer possible allele combinations with NGS. Eleven cases demonstrated polymorphism in exons outside the PBRs. Of these, only 2 cases showed no typing ambiguity with NGS. In the remaining 9 cases, though polymorphisms could be resolved in the PBR, ambiguities remained in exon 3, specifically at codon 96. In these cases, NGS could not distinguish lysine (K) from arginine (R). For example, DPB1*835:01 with the 96R motif could not be distinguished from DPB1*34:01with the 96K motif. The current investigation found that NGS cannot resolve all ambiguities at position 96, likely due, at least in part, to persistent cis-trans ambiguities. Our results suggest that although NGS drastically limited the number of ambiguities in 9 of 11 cases, it still could not adequately distinguish all immunologically relevant alleles. Inability to resolve the 96R/K epitope, which has been shown to be immunogenic, could impact solid organ transplantation, potentially leading to rejection in mismatched recipient-donor pairs at this position. Sequencing methods that generate longer reads, such as nanopore, may help resolve some of these cases if they are indeed due to cis-trans ambiguities.

Mesenchymal stem/stromal cells (MSCs) derived from placental tissue show great therapeutic potential and have been used in medical treatment, but the similarity and differences between the MSCs derived from various parts of the placenta remain unclear. In this study, we compared MSCs derived from different perinatal tissues, including the umbilical cord (UC), amniotic membrane (AM), chorionic plate (CP) and decidua parietalis (DP). Using human leukocyte antigen (HLA) typing and karyotype analysis, we found that the first three cell types were derived from the foetus, while the MSCs from the decidua parietalis were derived from the maternal portion of the placental tissue. Our results indicate that both foetal and maternal MSCs share a similar phenotype and multi-lineage differentiation potential, but foetal MSCs show a significantly higher expansion capacity than do maternal MSCs. Furthermore, MSCs from all sources showed significant differences in the levels of several paracrine factors.

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99–100% four-digit typing accuracy for both class I and II HLA genes, taking only ∼3 min to process a 30 × whole-genome BAM file on a desktop computer.

Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.

ObjectivesDrug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, delayed hypersensitivity reaction (DHR). We observed DRESS to inhibitors of interleukin 1 (IL-1) or IL-6 in a small group of patients with Still’s disease with atypical lung disease. We sought to characterise features of patients with Still’s disease with DRESS compared with drug-tolerant Still’s controls. We analysed human leucocyte antigen (HLA) alleles for association to inhibitor-related DHR, including in a small Kawasaki disease (KD) cohort.MethodsIn a case/control study, we collected a multicentre series of patients with Still’s disease with features of inhibitor-related DRESS (n=66) and drug-tolerant Still’s controls (n=65). We retrospectively analysed clinical data from all Still’s subjects and typed 94/131 for HLA. European Still’s-DRESS cases were ancestry matched to International Childhood Arthritis Genetics Consortium paediatric Still’s cases (n=550) and compared for HLA allele frequencies. HLA association also was analysed using Still’s-DRESS cases (n=64) compared with drug-tolerant Still’s controls (n=30). KD subjects (n=19) were similarly studied.ResultsStill’s-DRESS features included eosinophilia (89%), AST-ALT elevation (75%) and non-evanescent rash (95%; 88% involving face). Macrophage activation syndrome during treatment was frequent in Still’s-DRESS (64%) versus drug-tolerant Still’s (3%; p=1.2×10−14). We found striking enrichment for HLA-DRB1*15 haplotypes in Still’s-DRESS cases versus INCHARGE Still’s controls (p=7.5×10-13) and versus self-identified, ancestry-matched Still’s controls (p=6.3×10−10). In the KD cohort, DRB1*15:01 was present only in those with suspected anakinra reactions.ConclusionsDRESS-type reactions occur among patients treated with IL-1/IL-6 inhibitors and strongly associate with common HLA-DRB1*15 haplotypes. Consideration of preprescription HLA typing and vigilance for serious reactions to these drugs are warranted.